MIT7_13f08_lab07_Protocol_Dephosphorylating

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MIT OpenCourseWare http://ocw.mit.edu 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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7 . 1 3 F a l l 2 0 0 8 P a g e | 1 Dephosphorylating with CIP and Ligation Reactions 1. Preventing Vector Recircularization using Calf Intestinal Alkaline Phosphatase: Alkaline phosphatase catalyzes the removal of 5´ phosphate groups from DNA, RNA, ribo- and deoxyribonucleoside triphosphates. Since CIP-treated fragments lack the 5´phosphoryl termini required by ligases, they cannot self-ligate (1). This property can be used to decrease the vector background in cloning strategies. DNA ligase requires that one of the two pieces of DNA to be joined have a 5’-phosphate (donor). Vector DNA cut with a single enzyme will have two 5’-phosphate ends and two 3’ hydroxyl ends. Since they are on the same molecule, there is nothing to stop these from rejoing during a ligation. In fact, this reaction will proceed quite well, since it is essentially unimolecular. In order to minimize this reaction and favor ligation of the insert DNA, one can treat the digested vector DNA with Alkaline Phosphatase prior to purification. This enzyme will remove the 5’-phosphate groups. Since the insert will still
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